Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. 2011). Transfer of viral antigen specific T cells is a well-established procedure used for the treatment of transplant associated viral infections and rare viral-related malignancies. Similarly, isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma.
Novel specificities in T cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and heavy variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T cell cytotoxicity, however, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules including CD28, OX-40 (CD134), ICOS and 4-1BB (CD137) have been added alone (second generation) or in combination (third generation) to enhance survival and increase proliferation of CAR modified T cells. CARs have successfully allowed T cells to be redirected against antigens expressed at the surface of tumor cells from various malignancies including lymphomas and solid tumors (Jena, Dotti et al. 2010). However, for example, cancer cells are unstable and some cells may no longer possess the target antigen. These cells, referred to as antigen loss escape variants, escape destruction by the therapy and may continue to grow and spread unchecked. Cancer and healthy cells may express the same antigen although at different levels. In such case, having the possibility to combine at least two antigens in order for the engineered T cell to discriminate between healthy tissue and cancer cells would present extremely valuable advantage over actual technology for therapeutic purposes. Bispecific tandem CAR has already been described (International application: WO2013123061, US. Patent application: US20130280220). However, in this design the bispecific chimeric antigen receptor comprises (a) at least two antigen-specific targeting regions, (b) an extracellular spacer domain, (c) a transmembrane domain, (d) at least one co-stimulatory domain and (e) an intracellular signaling domain, wherein each antigen-specific targeting region comprises an antigen-specific single chain Fv (scFv) fragment, and binds a different antigen. Such design may theoretically still lead to the T-cell activation independently to the recognition and binding of both antigens as one cannot exclude that the binding of one single chain Fv may trigger activation. Kloss, Condomines et al. 2013 described another combinatorial antigen recognition approach. A CAR comprising a signaling domain mediated the recognition of one antigen and another receptor comprising a co-stimulatory domain specific for a second antigen are expressed at the surface of a T cell. This dual targeting approach facilitates augmented T cell reactivity against tumor positive for two antigens. However this approach alone fails to prevent T cell reactivity to single-positive tumors. To remedy this failure, search of adapted configuration of CAR are required.
To avoid the tuning of CAR used for the combinatorial antigen recognition, the inventors developed a system wherein activation of T cell is only induced through the combination of at least two signals. Each input signal alone does not induce the activation of T cell. Environmental signal integration by a modular AND gate within a CAR design may provide the ultimate strategy to insure safety and expand the number of surface antigens available for therapeutic purposes.
Logic gates are the basic building blocks in electronic circuits that perform logical operations. These have input and output signals in the form of 0's and 1's; ‘0’ signifies the absence of signal while ‘1’ signifies its presence. Similar to the electronic logic gates, cellular signals can serve as logic gates.
Synthetic biology applies many of the principles of engineering to the field of biology in order to create biological devices which can ultimately be integrated into increasingly complex systems. These principles include standardization of parts, modularity, abstraction, reliability, predictability, and uniformity (Andrianantoandro, Basu et al. 2006). The application of engineering principles to biology is complicated by the inability to predict the functions of even simple devices and modules within the cellular environment. Some of the confounding factors are gene expression noise, mutation, cell death, undefined and changing extracellular environments, and interactions with the cellular context (Andrianantoandro, Basu et al. 2006). Thus, while synthetic biology offers much promise in developing systems to address challenges faced in the fields of manufacturing, environment and sustainability, and health and medicine, the realization of this potential is currently limited by the diversity of available parts and effective design frameworks (Wang, Wei et al. 2013).